Clinical laboratory tests are routinely performed on the serum or plasma of whole blood. In a routine assay, red blood cells are separated from plasma by centrifugation, or red blood cells and various plasma proteins are separated from serum by clotting prior to centrifugation.
Haemoglobin (Hb), bilirubin (BR), biliverdin (BV) and light-scattering substances like lipid particles are typical substances which will interfere with, and affect spectrophotometric and other blood analytical measurements. Such substances are referred to as interferents. Elevated Hb in the blood, haemoglobinemia, can be due to disease states and as a result of specimen handling. Elevated bile pigments, namely BR and BV, can be due to disease states. Increased lipid particles in the blood, also known as hyperlipidemia or lipemia, can be due to disease states and dietary conditions. Lipemia is the major cause of turbidity in serum and plasma, and therefore the terms lipemia and turbidity are frequently used interchangeably.
Many tests conducted on plasma or serum samples employ a series of reactions which terminate after the generation of chromophores which facilitate detection by spectrophotometric measurements at one or two wavelengths. Measurement of interfering substances prior to conducting such tests is important in providing meaningful and accurate test results. In fact if a sample is sufficiently contaminated with interferents, tests are normally not conducted as the results will not be reliable.
Another set of potential interferents is blood substitutes. Blood substitutes are new products which are under development, for use instead of whole blood or red blood cells for transfusion. Blood transfusion is a life-saving process performed after severe blood loss during trauma or surgery. Some advantages of using a blood substitute instead of blood or red blood cells are as follows: 1. blood substitutes are expected to be universally compatible with all blood types, therefore cross-matching will not be necessary; 2. maximum storage time of blood is 42 days, whereas blood substitutes could have a much longer shelf-life; 3. purification a blood substitute may include heat treatment, which may eliminate the threat of hazardous viruses such as HIV. However, a challenge blood substitutes will pose to the clinical laboratory is managing the effects of blood substitutes on blood tests. Some blood substitutes will cause serum or plasma specimens to appear as whole blood or severely hemolyzed serum or plasma, hence the term pseudohemolysis may be used to describe such specimens. Hemolysis, the liberation of Hb from red blood cells into serum or plasma, may account for about 2 g/L, but blood substitutes can account for as much as 30 g/L of cross-linked Hb (CLHb), in a patient treated for severe blood loss. However, true hemolysis will not only make serum and plasma specimens appear red, but high concentrations of certain analytes inside red cells, e.g. potassium, will elevate the concentration of analytes in a serum or plasma specimen. Therefore, the effect of Hb-based blood substitutes on blood test results, is more predictable than the effect of true hemolysis.
Current methods used for detecting haemoglobinemia, bilirubinemia and lipemia or turbidity utilize visual inspection of the specimen with or without comparison to a coloured chart.
Most blood substitutes under development are made from human Hb, but another type of blood substitute has been reported which is a milky-white emulsion containing tiny beads of perfluorocarbons wrapped in a surfactant. For example, such perfluorocarbons are described in R. G. Pratt et al. “Quantitation of perfluorocarbon blood substitutes in tissues using F-19 magnetic resonance spectroscopy” Biomaterials, Artificial Cells and Immobilization Technology, Vol. 20, No. 2–4, 1992, pages 921–924, XP002057067.
The former will create pseudohemolysis while the latter will create pseudolipemia, in serum and plasma specimens. Subunits of Hb-based blood substitute are chemically cross-linked for stability (CLHb) and produce absorbance spectra which are very similar to the absorbance spectra of normal hemoglobin (Hb). A method and apparatus for determining, by absorption spectrophotometry, an analyte, such as bilirubin or hemoglobin, in human serum, in the presence of unknown interferents is described in European Patent Application 0 158 506. However, the method relies upon wet chemistry and dry chemistry assays in order to make the analyte determinations.
As such, currently there is no method for rapidly adjusting blood test results which are effected by blood substitutes. The present invention describes such a method. The method and apparatus of the present invention for measuring the concentration of blood substitutes in the presence of Hb, BR, BV and turbidity: The results of measurements of blood substitutes obtained are used to correct blood test results affected by the blood substitutes. Further the levels of other interferents can be measured in the presence of the blood substitutes. Therefore, true hemolysis can be measured in the presence of blood substitutes which cause “pseudo-hemolysis”.